Separation of postprandial lipoproteins: improved purification of chylomicrons using an ApoB100 immunoaffinity method

Grace Marie Jones,Russell Caccavello,Sergiu P Palii,Clive R Pullinger,John P Kane,Kathleen Mulligan,Alejandro Gugliucci,Jean-Marc Schwarz

doi:10.1194/jlr.d119000121

Abstract

Elevated levels of triglyceride-rich lipoproteins (TRLs), both fasting and postprandial, are associated with increased risk for atherosclerosis. However, guidelines for treatment are defined solely by fasting lipid levels, even though postprandial lipids may be more informative. In the postprandial state, circulating lipids consist of dietary fat transported from the intestine in chylomicrons (CMs; containing ApoB48) and fat transported from the liver in VLDL (containing ApoB100). Research into the roles of endogenous versus dietary fat has been hindered because of the difficulty in separating these particles by ultracentrifugation. CM fractions have considerable contamination from VLDL (purity, 10%). To separate CMs from VLDL, we produced polyclonal antibodies against ApoB100 and generated immunoaffinity columns. TRLs isolated by ultracentrifugation of plasma were applied to these columns, and highly purified CMs were collected (purity, 90-94%). Overall eight healthy unmedicated adult volunteers (BMI, 27.2 ± 1.4 kg/m2; fasting triacylglycerol, 102.6 ± 19.5 mg/dl) participated in a feeding study, which contained an oral stable-isotope tracer (1-13C acetate). We then used this technique on plasma samples freshly collected during an 8 h human feeding study from a subset of four subjects. We analyzed fractionated lipoproteins by Western blot, isolated and derivatized triacylglycerols, and calculated fractional de novo lipogenesis. The results demonstrated effective separation of postprandial lipoproteins and substantially improved purity compared with ultracentrifugation protocols, using the immunoaffinity method. This method can be used to better delineate the role of dietary sugar and fat on postprandial lipids in cardiovascular risk and explore the potential role of CM remnants in atherosclerosis.

Highlights

Elevated levels of triglyceride-rich lipoproteins (TRLs), both fasting and postprandial, are associated with increased risk for atherosclerosis
Dietary sugars, fructose, have been shown to increase TAG with supportive evidence that hepatic conversion of dietary sugars to Abbreviations: AUC, area under the curve; CM, chylomicron; CMIA, chylomicron purified from triglyceride-rich lipoprotein by immunoaffinity; CMU, chylomicron fraction isolated by ultracentrifugation; CMU+IA, chylomicron purified from CMU by immunoaffinity; DNL, de novo lipogenesis; FT, flow-through; MIDA, mass isotopomer distribution analysis; NFDM, nonfat dried milk; TAG, triacylglycerol; TRL, triglyceride-rich lipoprotein; VLDL obtained by ultracentrifugation (VLDLU), VLDL isolated by ultracentrifugation
Fractional DNL in TRL, CMU, and VLDLU To assess previously reported intestinal DNL in CMU and to understand the contribution of contamination by VLDL, we measured and compared fractional DNL in the CMU, TRL, and VLDL obtained by ultracentrifugation (VLDLU) fractions, which demonstrates the inability of ultracentrifugation to purify CMs from VLDL and supports the use our immunoaffinity method to possibly quantify enteral DNL

Summary

Introduction

Elevated levels of triglyceride-rich lipoproteins (TRLs), both fasting and postprandial, are associated with increased risk for atherosclerosis. Ultracentrifugation can achieve separation of triglyceride-rich lipoproteins (TRLs) and partial separation of CMs from VLDL, but cannot fully separate particles of overlapping densities, such as small CMs or CM remnants from large VLDL particles and VLDL remnants [12] This technical barrier has limited investigations that aim to characterize: 1) the respective contribution of TAG from the intestines and from the liver to postprandial lipid metabolism; 2) the kinetic aspects, namely the respective turnover rates of CMs versus VLDL particles; 3) the differential clearance rates of TAG; or 4) the impact of dietary factors, such as carbohydrates, sugar, and branched-chain amino acids, among others, on postprandial lipid metabolism and cardiovascular risk. In the second part of this study, we further characterized these purified particles; and to illustrate the application of our method, we measured fractional DNL [15] in the purified CM fractions using GC/MS and mass isotopomer distribution analysis (MIDA) [16,17,18,19] from samples collected from human volunteers during an 8 h feeding study

Methods

Results

Conclusion